Supplementary Material for: Effects of Cacna1d D307G mutation on blood pressure and kidney function in rats with salt loading

Introduction: Our recent findings revealed that CACNA1D D307G mutation participates in the early onset hypertension. Methods: we used CRISPR/Cas9 technique to generate the Cacna1d D307G mutation rat model and investigated the effects of Cacna1d D307G mutation on blood pressure (BP) and renal function. Rats fed normal-salt diet (NSD) had normal plasma aldosterone levels but higher plasma ET-1 and mildly elevated systolic blood pressure (SBP) in D307G and G307G rats compared with the wild type (WT) until 24 weeks. Renal function and renal histopathology did not significantly differ among the three groups. Results: When fed high-salt diet (HSD), D307G and G307G rats showed more sensitivity to HSD. The results showed a further increase in SBP than in WT rats. Plasma and vascular ET-1 level and cortex and renal artery endothelin type A receptor (ETA) protein expression were significantly increased. Enhanced renal injury was also noted as indicated by an increased ratio of kidney weight/body weight, elevated urinary protein and albumin/creatinine ratio, higher kidney injury molecule-1 (KIM-1) levels, advanced fibrosis and apoptosis, and inflammation. Further experiments revealed a reduction in urinary sodium excretion and creatinine clearance. Higher protein expression of renal cortex epithelial sodium channel α subunit (αENaC) was confirmed in D307G and G307G rats fed HSD. However, a selective ETA receptor blockade (ABT-627) could partially reverse the increased SBP, increased serum KIM-1 level, upregulated renal cortex protein expression of αENaC, and reduced urinary sodium excretion with reduced creatinine clearance in D307G rats fed HSD. Conclusion: Activation of ET-1/ETA system in D307G mutation rats might contributed to increased sensitivity to salt loading, augmented hypertension, and exacerbated the renal injury.

引言：近期的研究成果揭示了CACNA1D D307G突变在高血压早期发病机制中的作用。研究方法：本研究采用CRISPR/Cas9技术构建了Cacna1d D307G突变大鼠模型，并探讨了Cacna1d D307G突变对血压（BP）和肾脏功能的影响。喂以正常盐分饮食（NSD）的大鼠具有正常的血浆醛固酮水平，但与野生型（WT）相比，D307G和G307G大鼠的血浆ET-1水平较高，且收缩压（SBP）轻微升高，这一现象持续至24周。三组大鼠的肾功能和肾脏组织病理学无显著差异。结果：在高盐分饮食（HSD）喂养条件下，D307G和G307G大鼠对HSD的敏感性增强，其SBP的升高程度超过了WT大鼠。血浆和血管ET-1水平以及皮质和肾动脉内皮素A受体（ETA）蛋白表达显著增加。肾脏损伤也得到增强，这体现在肾脏重量与体重比的增加、尿蛋白和尿肌酐比率的升高、肾脏损伤分子-1（KIM-1）水平的上升、纤维化和凋亡的进展，以及炎症的发生。进一步的实验揭示了尿钠排泄减少和肌酐清除率降低。在高盐分饮食喂养的D307G和G307G大鼠中，肾皮质上皮钠通道α亚单位（αENaC）蛋白表达量升高得到证实。然而，选择性ETA受体阻断剂（ABT-627）能够部分逆转D307G大鼠在高盐分饮食喂养条件下SBP的升高、血清KIM-1水平的增加、肾皮质αENaC蛋白表达的升高，以及尿钠排泄减少和肌酐清除率降低。结论：ET-1/ETA系统在D307G突变大鼠中的激活可能导致了盐负荷敏感性的增加、高血压的加剧以及肾脏损伤的恶化。